Despite considerable advances in our understanding of the etiology of arteriosclerosis, about 30% cardiovascular disease (CVD) cannot be explained by traditional risk factors. Hyperhomocysteinemia (HHcy) is an important non-lipid CVD risk factor for which the underlying mechanism is unknown. We have previously proposed that Hcy promotes atherosclerosis by stimulating vascular smooth muscle cell proliferation and by inhibiting endothelial cell growth. To address the role of HHcy in an atherogenic background, we recently generated double knockout (KO) mice with targeted deletions of the genes for apolipoprotein E (apoE) and cystathionine b-synthase (CBS), which converts Hcy to cystathionine. Deletion of the CBS gene in apoE-/- mice resulted severe HHcy, which was associated with increased aortic lesions even in the absence of dietary manipulation. We therefore hypothesize that Hcy is a causative factor of atherosclerosis and causes macrophage activation and endothelial dysfunction. This project will study these hypothesis utilizing four linked specific aims. First, in Aim 1, experiments will evaluate HHcy atherosclerosis by examining early disease in aortic sinus in CBS/apoE double KO mice, and by studying post-injury atherosclerosis in CBS KO mice. Second, in Aim 2, studies are proposed to determine the effect of HHcy on macrophage function by bone marrow transplantation and by macrophage phagocytosis assay in the double KO mice. Third, in Aim 3, studies will characterize HHcy endothelial dysfunction and identify the mechanistic basis. Finally, in Aim 4, experiments will study atherosclerosis, functions of macrophage and endothelium following HHcy lowering therapy. We believe that the overall integrative approaches proposed will provide valuable new information to establish the links between Hcy and atherosclerosis.